The overall goal of this project is to understand how molecules are sorted to maintain the distinct protein and lipid compositions of organelles. The focus is on endocytic sorting, but endocytic pathways intersect with vesicular and non-vesicular transport pathways involving other organelles, including the ER and the Golgi. Methods have been developed to characterize endocytic pathways in cells morphologically and kinetically, including measurements of kinetics and efficiency of many sorting steps. Several proteins have been shown to play some role in sorting, but in many cases the precise role remains uncertain. The proposed work will test specific roles in well-defined trafficking steps for several key sorting proteins. The methods used will include mutation of binding sequences in cargo proteins, knockdown of sorting proteins by RNAi, and rapid inactivation by microinjection of inhibitors, chemically-induced crosslinking, or photophysical inactivation. Effects on trafficking will be measured by well-characterized quantitative optical microscopy assays. Cargo proteins to be studied include transferrin receptors, the cation-independent mannose-6-phosphate receptor, furin, and TGN38. The sorting proteins to be examined include clathrin, adaptins, GGAs, and the retromer complex. Protein traffic is intimately linked with lipid traffic and cholesterol levels. Novel methods have been developed to directly observe sterol trafficking in living cells, and these will be used to analyze the mechanisms for vesicular and non-vesicular sterol traffic. The role of the StAR family of proteins in nonvesicular sterol transport will be evaluated. The effects of changes in expression levels of various ABC transporters and stearoyl-coA-desaturase on the distribution and trafficking of sterol in cells will be measured. Changes in the biophysical properties of cell membranes under these conditions will be analyzed by fluorescence and by ESR of spin-labeled lipids. A novel mechanism by which unsaturated fatty acids alter the trafficking of the sterol response element binding protein will be characterized.